Stroke remains the third leading cause of death in the US, behind heart disease and cancer. Each year 750,000 Americans suffer a diagnosed stroke and this number will double by 2050, given the advancing average age of the population, barring further developments in either prevention and/or treatment. The goal of our proposed set of projects is to critically evaluate astrocytes as a potential new therapeutic target in stroke. Over the past few years, a virtual revolution has occurred in our understanding of the cell biology and physiology of astrocytes, and in our understanding of their interactions with neurons in the normal brain. Yet despite their obvious relevance to stroke, the contribution of astrocytes to the process of ischemic infarction has not yet been well-studied. In this application, we propose to use a multimodal approach to define the role of astrocytes in the pathogenesis of ischemic stroke. We shall make use of 2-photon laser scanning microscopy to visualize astrocytes labeled with GFP and Ca2+ sensitive dyes, both in slices and in the intact brain. We will also utilize GFP labeling under control of the astrocyte specific GFAP promoter, to sort astrocytes from animals with experimental stroke, so as to characterize ischemia-induced changes in gene expression. The GFAP promoter will be utilized to knock out expression of HIF-1alpha, the master regulator of ischemia-induced gene expression, and this approach will allow us to define how hypoxia-induced gene expression in astrocytes affect neuronal survival following experimental stroke. The program benefits from the very different training of its investigators, whose expertise spans electrophysiology, stroke models, Ca2+-imaging techniques, fluorescent activated cell sorting, and molecular genetic manipulation. Our hope is that these studies ultimately will result in new treatment options for patients with acute ischemic stroke.